Review on the role of antioxidant supplementation against oxidative stress: a human and animal approach to male fertility

Male infertility is one important factor among the multifactorial causes of couple infertility, being oxidative stress one of the main related sources. Sperm is a specialized cell extremely susceptible to stress. To understand and mitigate this event, many studies have used different antioxidants, orally or in vitro supplementation, trying to improve sperm quality and function. Considering the extensive available literature regarding approaches and attempts to solve male fertility issues, the aim of this review is evaluating the effects of antioxidant supplementation on sperm, in both humans and experimental models with animals. This review selected original data from PubMed. The keywords used were: antioxidant, sperm, male fertility, antioxidant supplementation, male infertility; and the term "rodents" was added to the descriptors “antioxidant” and “male fertility”. Only studies published in indexed journals, in English, between 2015 and 2019 were included. This review involves i) human sperm and ii) rodent sperm. For the human approach, the search retrieved 496 articles and 80 were included, among which 28 studies were of in vitro antioxidant supplementation, 19 involved oral antioxidant supplementation and the remaining 33 concerned quantification of oxidants and antioxidants already present in the seminal samples. For the rodent approach, 152 articles were retrieved and 52 were included: 3 of varicocele, 11 of diabetes, 10 of therapeutic drugs, 3 of physical exercise, 10 of environmental exposure and 3 of heat stress. The remaining studies involved oxidative stress status in experimental models. Antioxidants use for reproductive purposes is increasing in an attempt to achieve better gametes and embryos. Vitamins C, B and E, selenium and zinc are the most commonly used antioxidants, with remarkable evidences in improving pathophysiological seminal conditions.

Homozygous mutation in SLO3 leads to severe asthenoteratozoospermia due to acrosome hypoplasia and mitochondrial sheath malformations

Background Potassium channels are important for the structure and function of the spermatozoa. As a potassium transporter, the mSlo3 is essential for male fertility as Slo3 knockout male mice were infertile with the series of functional defects in sperm cells. However, no pathogenic variant has been detected in human SLO3 to date. Here we reported a human case with homozygous SLO3 mutation. The function of SLO3 in human sperm and the corresponding assisted reproductive strategy are also investigated. Methods We performed whole-exome sequencing analysis from a large cohort of 105 patients with asthenoteratozoospermia. The effects of the variant were investigated by quantitative RT-PCR, western blotting, and immunofluorescence assays using the patient spermatozoa. Sperm morphological and ultrastructural studies were conducted using haematoxylin and eosin staining, scanning and transmission electron microscopy. Results We identified a homozygous missense variant (c.1237A > T: p.Ile413Phe) in the sperm-specific SLO3 in one Chinese patient with male infertility. This SLO3 variant was rare in human control populations and predicted to be deleterious by multiple bioinformatic tools. Sperm from the individual harbouring the homozygous SLO3 variant exhibited severe morphological abnormalities, such as acrosome hypoplasia, disruption of the mitochondrial sheath, coiled tails, and motility defects. The levels of SLO3 mRNA and protein in spermatozoa from the affected individual were reduced. Furthermore, the acrosome reaction, mitochondrial membrane potential, and membrane potential during capacitation were also afflicted. The levels of acrosome marker glycoproteins and PLCζ1 as well as the mitochondrial sheath protein HSP60 and SLO3 auxiliary subunit LRRC52, were significantly reduced in the spermatozoa from the affected individual. The affected man was sterile due to acrosome and mitochondrial dysfunction; however, intra-cytoplasmic sperm injection successfully rescued this infertile condition. Conclusions SLO3 deficiency seriously impact acrosome formation, mitochondrial sheath assembly, and the function of K+ channels. Our findings provided clinical implications for the genetic and reproductive counselling of affected families.

Oxidative Stress Measurement in Frozen/Thawed Human Sperm: The Protective Role of an In Vitro Treatment with Myo-Inositol

Despite its widespread use, sperm cryopreservation induces serious detrimental alterations in sperm function; indeed, it is commonly associated with decreased sperm viability and motility, and DNA fragmentation. Mechanisms of human sperm cryodamage are thought to be multifactorial, but oxidative stress seems to have a prominent role. A huge amount of data supported the cryoprotective effect of different antioxidants able to minimize the detrimental effects of reactive oxygen species (ROS) and improve the quality of spermatozoa. Among others, myo-inositol is one of the most powerful and has been reported to be effective in improving sperm quality and motility when used both in vivo and in vitro. This study aimed to determine the in vitro impact of myo-inositol in ameliorating sperm oxidative status during sperm cryopreservation. In particular, we demonstrated a significant improvement of sperm parameters (vitality and motility) when myo-inositol was added after sperm thawing (p < 0.05). Moreover, we showed that myo-inositol induces a significant increase in oxygen consumption, the main index of oxidative phosphorylation efficiency and ATP production. Finally, by means of 2D-electrophoresis, we demonstrated a significant decrease in the level of carbonyl groups, the main structural changes occurring in conditions of oxidative stress (p < 0.05). In conclusion, the sperm cryopreservation procedure we developed, assuring the reduction of ROS-induced sperm modifications, may improve the in vitro procedure currently used in ART laboratory for sperm cryostorage.

Protein Kinase A (PRKA) Activity Is Regulated by the Proteasome at the Onset of Human Sperm Capacitation

The proteasome increases its activity at the onset of sperm capacitation due to the action of the SACY/PRKACA pathway; this increase is required for capacitation to progress. PRKA activity also increases and remains high during capacitation. However, intracellular levels of cAMP decrease in this process. Our goal was to evaluate the role of the proteasome in regulating PRKA activity once capacitation has started. Viable human sperm were incubated in the presence and absence of epoxomicin or with 0.1% DMSO. The activity of PRKA; the phosphorylation pattern of PRKA substrates (pPRKAs); and the expression of PRKAR1, PRKAR2, and AKAP3 were evaluated by Western blot. The localization of pPRKAs, PRKAR1, PRKAR2, and AKAP3 was evaluated by immunofluorescence. Treatment with epoxomicin changed the localization and phosphorylation pattern and decreased the percentage of pPRKAs-positive sperm. PRKA activity significantly increased at 1 min of capacitation and remained high throughout the incubation. However, epoxomicin treatment significantly decreased PRKA activity after 30 min. In addition, PRKAR1 and AKAP3 were degraded by the proteasome but with a different temporal kinetic. Our results suggest that PRKAR1 is the target of PRKA regulation by the proteasome.

Strategies and Perspectives for UV Resonance Raman Applicability in Clinical Analyses of Human Sperm RNA

Developing a deeper knowledge about the impact of DNA and RNA epigenetic mutations on sperm production and fertilization performance is essential for selecting best quality samples in Assisted Reproductive Technologies (ART). Indeed, sperm RNAs adenine and guanine are likely to be methylated in low quality RNA sperm samples and their study requires the employment of techniques able to isolate high quality nucleic acids. UV resonance Raman spectroscopy represents a valuable tool that is able to monitor peculiar molecular modifications occurring predominantly in nucleic acids, being less sensitive to the presence of other biological compounds. In this work, we used an UV Resonance Raman (UVRR) setup coupled to a synchrotron radiation source tuned at 250 nm, in order to enhance sperm RNAs adenine and guanine vibrational signals, reducing also the impact of a fluorescence background typically occurring at lower energies. Despite that our protocol should be further optimized and further analyses are requested, our results support the concept that UVRR can be applied for setting inexpensive tools to be employed for semen quality assessment in ART.